Alkaline cellulases and microorganisms capable of producing same

Abstract

Alkaline cellulases having an optimum pH in an alkaline range and being stable over a broad pH range are produced by microorganisms which belong to the genus Bacillus and grow in a neutral medium. These alkaline cellulases are rarely inhibited by ingredients ordinarily incorporated in detergents such as surface active agents, proteinases and chelating agents, so that they can be conveniently used in detergent compositions.

What is claimed is: 
     
       1. A substantially pure alkaline cellulase enzyme K-580 isolated from Bacillus species KSM-580 having the following enzymatic properties: (1) Action:   acting well on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose and causing them to be dissolved, thereby forming reducing sugars such as glucose;   (2) Substrate specificity:   having activity on carboxymethyl cellulose, cellulose powder, microcrystalline cellulose and filter paper;   (3) Working pH and optimum pH:   a working pH range from 3 to 12.5 and an optimum pH from 7 to 10 with a relative activity of not less than 50% of the activity at an optimum pH being shown in the range of 4.5 to 10.5;   (4) pH stability:   very stable and not inactivated at a pH of 4.5 to 12 and an activity of not less than about 50% being maintained at a pH of 3.5 to 12.5;   (5) Optimum temperature:   a working temperature in a wide range of 15 to 80° C. and an optimum temperature of 65° C.; in the range of 50° to 75° C., the activity is not less than 50% of the activity at the optimum temperature;   (6) Molecular weight;   peaks of the molecular weight are at about 18,000 and about 50,000 (when determined by gel filtration);   (7) Influences of metal ions;   inhibited by Hg 2  + and activated by Ba 2+ , Ca 2+ , Co 2+  and Cd 2+  ;   (8) Influences of surface active agents:   sodium linear alkylbenzenesulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium alpha-olefin sulfonates, sodium alpha-sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers rarely inhibit the activity;   (9) Proteinase resistance;   resistant to proteinases;   (10) Influences of chelating agents:   ethylenediamine tetraacetic acid, ethyleneglycolbis-(beta-aminoethylether)-N,N,N',N"-tetraacetic acid, citric acid, sodium tripolyphosphate and zeolite do not inhibit the activity.   
     
     
       2. A substantially pure alkaline cellulase enzyme K-425 isolated from Bacillus species KSM-425 having the following enzymatic properties: (1) Action:   acting well on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose and causing them to be dissolved, thereby forming reducing sugars such as glucose;   (2) Substrate specificity:   having activity on carboxymethyl cellulose, cellulose powder, microcrystalline cellulose, filter paper, p-nitrophenyl cellobioside and cellobiose;   (3) Working pH and optimum pH:   a working pH range of from 3.5 to 12.5 and an optimum pH of from 8 to 10 with a relative activity of not less than 50% of the activity at an optimum pH being shown in the range of 5.5 to 10.5;   (4) pH stability:   very stable and not inactivated at a pH of 5 to 11 and an activity of not less than about 50% being maintained at a pH of 3 to 12;   (5) Optimum temperature;   a working temperature in a wide range of 15 to 75° C. and an optimum temperature is 50° C.; in the range of 35° to 55° C., the activity is not less than 50% of the activity at the optimum temperature;   (6) Molecular weight:   about 35,000 (when determined by gel filtration);   (7) Influences of metal ions:   inhibited by Hg 2+  and Ba 2+  and activated by Co 2+  ;   (8) Influences of surface active agents:   sodium linear alkylbenzenesulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium alpha-olefin sulfonates, sodium alpha-sulfonated aliphatic acid esters, sodium alkylsulfonates, soap and polyoxyethylene secondary alkyl ethers rarely inhibit the activity;   (9) Proteinase resistance:   resistant to proteinases;   (10) Influences of chelating agents:   ethylenediamine tetraacetic acid, ethyleneglycolbis-(beta-aminoethylether)-N,N,N',N"-tetraacetic acid, citric acid, sodium tripolyphosphate and zeolite do not inhibit the activity.   
     
     
       3. A substantially pure alkaline cellulase enzyme K-521 isolated from Bacillus species KSM-521 having the following enzymatic properties: (1) Action:   acting well on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose and causing them to be dissolved, thereby forming reducing sugars such as glucose;   (2) Substrate specificity:   having activity on carboxymethyl cellulose, cellulose powder, microcrystalline cellulose, filter paper, p-nitrophenyl cellobiose and cellobiose;   (3) Working pH and optimum pH:   a working pH range of from 3 to 12.5 and an optimum pH of from 7 to 10 with a relative activity of not less than 50% of the activity at an optimum pH being shown in the range of 4.5 to 10.5;   (4) pH stability:   very stable and not inactivated at a pH of 5 to 12 and an activity of not less than about 50% being maintained at a pH of 4.5 to 12.5;   (5) Optimum temperature:   a working temperature in a wide range of 15 to 80° C. and an optimum temperature is 60° C.; in the range of 45° to 65° C., the activity is not less than 50% of the activity at the optimum temperature;   (6) Molecular weight:   about 31,000 (when determined by gel filtration);   (7) Influences of metal ions:   inhibited by Hg 2+  and activated by Ca 2+  ;   (8) Influences of surface active agents:   sodium linear alkylbenzenesulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium alpha-olefin sulfonates, sodium alpha-sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers rarely inhibit the activity;   (9) Proteinase resistance:   resistant to proteinases;   (10) Influences of chelating agents:   ethylenediamine tetraacetic acid, ethyleneglycolbis-(beta-aminoethylether)-N,N,N',N"-tetraacetic acid, citric acid, sodium tripolyphosphate and zeolite do not inhibit the activity.   
     
     
       4. A substantially pure alkaline cellulase enzyme K-522 isolated from Bacillus KSM-522 having the following enzymatic properties: (1) Action:   acting well on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose and causing them to be dissolved, thereby forming reducing sugars such as glucose;   (2) Substrate specificity:   having activity on carboxymethyl cellulose, cellulose powder, phosphoric acid-swollen cellulose, alkali-swollen cellulose, microcrystalline cellulose, filter paper, and p-nitrophenyl cellobioside;   (3) Working pH and optimum pH:   a working pH range of from 3 to 12.5 and an optimum pH of from 7 to 10 with a relative activity of not less than 50% of the activity at an optimum pH being shown in the range of 4.5 to 10.5;   (4) pH stability:   very stable and not inactivated at a pH of 5 to 12 and an activity of not less than about 50% being maintained at a pH of 4.5 to 12.5;   (5) Optimum temperature:   a working temperature in a wide range of 15° to 80° C. and an optimum temperature of 60° C.; in the range of 45° to 65° C., the activity is not less than 50% of the activity at the optimum temperature;   (6) Molecular weight:   about 35,000 (when determined by gel filtration);   (7) Influences of metal ions:   inhibited by Hg 2+  ;   (8) Influences of surface active agents:   sodium linear alkylbenzenesulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium alpha-olefin sulfonates, sodium alpha-sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers rarely inhibit the activity;   (9) Proteinase resistance:   resistant to proteinases;   (10) Influences of chelating agents:   ethylenediamine tetraacetic acid, ethyleneglycolbis-(beta-aminoethylether)-N,N,N',N"-tetraacetic acid, citric acid, sodium tripolyphosphate and zeolite do not inhibit the activity.   
     
     
       5. A biologically pure culture of Bacillus sp. KSM-580 deposited as FERM BP-1511 which is capable of producing the alkaline cellulase K-580 of claim 1. 
     
     
       6. A biologically pure culture of Bacillus sp. KSM-425 deposited as FERM BP-1505 which is capable of producing the alkaline cellulase K-425 of claim 2. 
     
     
       7. A biologically pure culture of Bacillus sp. KSM-521 deposited as FERM BP-1507 which is capable of producing the alkaline cellulase K-521 of claim 3. 
     
     
       8. A biologically pure culture of Bacillus sp. KSM-522 deposited as FERM BP-1512 which is capable of producing the alkaline cellulase K-522 of claim 4. 
     
     
       9. A process for producing an alkaline cellulase enzyme having an optimum pH not less than pH 8 which comprises cultivating an alkaline cellulase enzyme producing bacterium selected from the group consisting of Bacillus species KSM-580 deposited as FERM BP-1511, Bacillus species KSM-425 deposited as FERM BP-1505, Bacillus species KSM-521 deposited as FERM BP-1507 and Bacillus species KSM-522 deposited as FERM BP-1512, which is capable of growing in a neutral medium, in a neutral medium and collecting the alkaline cellulase enzyme having an optimum pH not less than pH 8 from the resulting culture product. 
     
     
       10. The process according to claim 9, wherein said alkaline cellulase has the following enzymatic properties: (1) optimum pH: 8-10,   (2) inhibited in the presence of Hg 2+ ,   (3) rarely inhibited with proteinases, surface active agents and chelating agents,   (4) the carboxymethyl cellulose decomposing activity (Cx activity) being a main activity with additional filter paper disintegrating activity and microcrystalline cellulose activity (C 1  activity).   
     
     
       11. The process according to claim 9, wherein the bacterium is an alkaline cellulase K-580-producing bacterium Bacillus sp. KSM-580 deposited as FERM BP-1511. 
     
     
       12. The process according to claim 9, wherein the bacterium is an alkaline cellulase K-425-producing bacterium Bacillus sp. KSM-425 deposited as FERM BP-1505. 
     
     
       13. The process according to claim 9, wherein the bacterium is an alkaline cellulase K-521-producing bacterium Bacillus sp. KSM-521 and deposited as FERM BP-1507. 
     
     
       14. The process according to claim 9, wherein the bacterium is an alkaline cellulase K-522-producing bacterium Bacillus sp. KSM-522 deposited as FERM BP-1512.